A vast number of new drug targets are now being identified using a combination of genomics, bioinformatics, genetics, and high-throughput (HTP) biochemistry. Genomics provides information on the genetic composition and the activity of an organism""s genes. Bioinformatics uses computer algorithms to recognize and predict structural patterns in DNA and proteins, defining families of related genes and proteins. The information gained from the combination of these approaches is expected to boost the number of drug targets, usually proteins, from the current 500 to over 10,000 in the coming decade.
The number of chemical compounds available for screening as potential drugs is also growing dramatically due to recent advances in combinatorial chemistry, the production of large numbers of organic compounds through rapid parallel and automated synthesis. The compounds produced in the combinatorial libraries being generated will far outnumber those compounds being prepared by traditional, manual means, natural product extracts, or those in the historical compound files of large pharmaceutical compares.
Both the rapid increase of new drug targets and the availability of vast libraries of chemical compounds creates an enormous demand for new technologies which improve the screening process. Current technological approaches which attempt to address this need include multiwell-plate-based screening systems, cell-based screening systems, microfluidics-based screening systems, and screening of soluble targets against solid-phase synthesized drug components.
Automated multiwell formats are the best developed high-throughput screening systems. Automated 96-well plate-based screening systems are the most widely used. The current trend in plate-based screening systems is to reduce the volume of the reaction wells further, thereby increasing the density of the wells per plate (96-well to 384-, and 1536-well per plate). The reduction in reaction volumes results in increased throughput, dramatically decreased bioreagent costs, and a decrease in the number of plates which need to be managed by automation.
However, although increases in well numbers per plate are desirable for high throughput efficiency, the use of volumes smaller than 1 microliter in the well format generates significant problems with evaporation, dispensing times, protein inactivation, and assay adaptation. Proteins are very sensitive to the physical and chemical properties of the reaction chamber surfaces. Proteins are prone to denaturation at the liquid/solid and liquid/air interfaces. Miniaturization of assays to volumes smaller than 1 microliter increases the surface to volume ratio substantially. (Changing volumes from 1 microliter to 10 nanoliter increases the surface ratio by 460%, leading to increased protein inactivation.) Furthermore, solutions of submicroliter volumes evaporate rapidly, within seconds to a few minutes, when in contact with air. Maintaining microscopic volumes in open systems is therefore very difficult.
Other types of high-throughput assays, such as miniaturized cell-based assays are also being developed. Miniaturized cell-based assays have the potential to generate screening data of superior quality and accuracy, due to their in vivo nature. However, the interaction of drug compounds with proteins other than the desired targets is a serious problem related to this approach which leads to a high rate of false positive results.
Microfluidics-based screening systems that measure in vitro reactions in solution make use often to several-hundred micrometer wide channels. Micropumps, electroosmotic flow, integrated valves and mixing devices control liquid movement through the channel network. Microfluidic networks prevent evaporation but, due to the large surface to volume ratio, result in significant protein inactivation. The successful use of microfluidic networks in biomolecule screening remains to be shown.
Drug screening of soluble targets against solid-phase synthesized drug components is intrinsically limited. The surfaces required for solid state organic synthesis are chemically diverse and often cause the inactivation or non-specific binding of proteins, leading to a high rate of false-positive results. Furthermore, the chemical diversity of drug compounds is limited by the combinatorial synthesis approach that is used to generate the compounds at the interface. Another major disadvantage of this approach stems from the limited accessibility of the binding site of the soluble target protein to the immobilized drug candidates.
DNA microarray technology is not immediately transferable to protein screening microdevices. To date, microarrays are exclusively available for nucleic acid hybridization assays (xe2x80x98DNA-chipsxe2x80x99). Their underlying chemistry and materials are not readily transferable to protein assays. Nucleic acids withstand temperatures up to 100xc2x0 C., can be dried and re-hydrated without loss of activity and bound directly to organic adhesion layers absorbed on surfaces such as glass. In contrast, proteins must remain hydrated, kept at ambient temperatures, and are very sensitive to the physical and chemical properties of the support materials. Therefore, maintaining protein activity at the liquid-solid interface requires entirely different immobilization strategies than those used for nucleic acids. Additionally, the proper orientation of the protein at the interface is desirable to ensure accessibility of their active sites with interacting molecules.
In addition to the goal of achieving high-throughput screening of compounds against targets to identify potential drug leads, researchers also need to be able to identify a highly specific lead compound early in the drug discovery process. Analyzing a multitude of members of a protein family or forms of a polymorphic protein in parallel enables quick identification of highly specific lead compounds. Proteins within a structural family share similar binding sites and catalytic mechanisms. Often, a compound that effectively interferes with the activity of one family member also interferes with other members of the same family. Using standard technology to discover such additional interactions requires a tremendous effort in time and costs and as a consequence is simply not done.
However, cross-reactivity of a drug with related proteins can be the cause of low efficacy or even side effects in patients. For instance, AZT, a major treatment for AIDS, blocks not only viral polymerases, but also human polymerases, causing deleterious side effects. Cross-reactivity with closely related proteins is also a problem with nonsteroidal anti-inflammatory drugs (NSAIDs) and aspirin. These drugs inhibit cyclooxygenase-2, an enzyme which promotes pain and inflammation. However, the same drugs also strongly inhibit a related enzyme, cyclooxygenase-1, that is responsible for keeping the stomach lining and kidneys healthy, leading to common side-effects including stomach irritation.
For the foregoing reasons, there is a need for miniaturized devices and for methods for the parallel, in vitro, high-throughput screening of functionally and/or structurally related protein targets against potential drug compounds in a manner that minimizes reagent volumes and protein inactivation problems.
The present invention is directed to a device and methods of use of the device that satisfy the need for parallel, in vitro, high-throughput screening of functionally or structurally related protein targets against potential drug, compounds in a manner that minimizes reagent volumes and protein inactivation problems.
One embodiment of the present invention provides a device that has a plurality of noncontiguous reactive sites and is useful for processing fluid samples. On each of the reactive sites of the device, biological moieties are immobilized on a monolayer via an affinity tag which enhances the site-specific immobilization of the biological moiety onto the monolayer. Each of the reactive sites is separated from neighboring reactive sites by substrate that is free of the monolayer. The monolayer is on, a portion of a surface of a substrate and comprises molecules of the formula X-R-Y where R is a spacer, X is a functional group that binds R to the surface, and Y is a functional group for binding a biological moiety onto the monolayer via the affinity tag. Each of the reactive sites is displayed on the device in a manner that allows it to react with a component of a fluid sample.
In a preferred embodiment the device of the present invention is a micromachined or microfabricated device.
In a particularly preferred embodiment of the device, the plurality of reactive sites are contained within parallel microchannels. These microchannels may be microfabricated into or onto the substrate.
Optionally, at least one coating may be formed on the substrate or applied to the substrate of a device of the present invention such that the coating is positioned between the substrate and the monolayer of each reactive site.
The monolayer of a device of the present invention may optionally be a mixed monolayer of more than one type of organic molecule.
In a preferred embodiment, an adaptor molecule is also included in the device of the present invention to link the affinity tag to the biological moiety.
The affinity tag, biological moiety, and the adaptor molecule (if present) are preferably, but not necessarily, a fusion protein.
The biological moiety immobilized on one reactive site can either be the same as or different from the biological moiety immobilized on a second reactive site. If the reactive sites are different, the biological moieties of the different reactive sites are preferably members of the same protein family or are otherwise functionally or structurally related.
The present invention further provides for methods of using the device to screen a plurality of biological moieties in parallel for their ability to interact with a component of a fluid sample. The interaction being assayed may be a binding interaction or a catalytic one. Some embodiments of these methods first involve delivering the fluid sample to the reactive sites of the device. If binding between the biological moiety and the component is to be detected, the reactive sites are then optionally washed to remove any unbound component from the area. If binding interactions are being monitored, the methods also involve detecting, either directly or indirectly, the retention of the component at each reactive site. If the interaction being assayed is catalytic, then the presence, absence, or amount of reaction product is instead detected.
In other embodiments of the present invention, similar methods are used diagnostically to screen a fluid sample for the presence, absence, or amount of a plurality of analytes (in parallel).
In another method, the device may also be used to screen a plurality of drug candidates in parallel for their ability to bind or react with a biological moiety. In this method, different fluid samples, each containing a different drug candidate (or a different mixture of drug candidates) to be tested, is delivered to the different reactive sites of the invention device.
The present invention also provides for methods of determining in parallel whether or not a plurality of proteins belong to a certain protein family based on either binding to a common ligand or reactivity with a common substrate. These methods involve delivering a fluid sample comprising a ligand or substrate of a known protein family to the reactive sites of the invention device that contain the different proteins and then detecting, either directly or indirectly, for binding or reaction with the known ligand that is characteristic of the protein family.
An alternative embodiment of the invention provides a device for processing a fluid sample that comprises a substrate, a plurality of parallel microchannels microfabricated into or onto said substrates and a moiety immobilized within at least one of the parallel microchannels, in such a way that the moiety interacts with a component of the fluid sample. In a preferred embodiment, the immobilized moiety is a biological moiety such as a protein.